The binding of FKBP23 to BiP modulates BiP's ATPase activity with its PPIase activity

Peptidyl-prolyl cis-trans-isomerases (PPIases) are enzymes that can cis-trans-isomerize a Xaa-Pro peptide bond. Three families of PPIases are known: cyclophilins, FKBPs, and parvulins. The physiological functions of the PPIases are only poorly understood. In previous work, we reported that the mouse FK506-binding protein 23 (mFKBP23), which comprises an N-terminal PPIase domain and a C-terminal domain with Ca(2+)-binding sites, binds to mBiP in the endoplasmic reticulum (ER) and this binding is affected by the Ca(2+) concentration. In this study, we demonstrate the ability of mFKBP23 to modulate the ATPase activity of BiP, and that the bound mFKBP23, but not the free mFKBP23, can suppress the ATPase activity of mBiP through its PPIase activity.     

The chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains

Cyclophilin 40 (CyP40), an immunophilin cochaperone present in steroid receptor-Hsp90 complexes, contains an N-terminal peptidylprolyl isomerase (PPIase) domain separated from a C-terminal Hsp90-binding tetratricopeptide repeat (TPR) domain by a 30-residue linker. To map CyP40 chaperone function, CyP40 deletion mutants were prepared and analysed for chaperone activity. CyP40 fragments containing the PPIase domain plus linker or the linker region and the adjoining TPR domain retained chaperone activity, whilst individually, the catalytic and TPR domains were devoid of chaperoning ability. CyP40 chaperone function then, is localized within the linker that forms a binding cleft with potential to accommodate non-native substrates.     

1.88 A crystal structure of the C domain of hCyP33: a novel domain of peptidyl-prolyl cis-trans isomerase

Cyclophilins (CyPs) are a widespreading protein family in living organisms and possess the activity of peptidyl-prolyl cis-trans isomerase (PPIase), which is inhibited by cyclosporin A (CsA). The human nuclear cyclophilin (hCyP33) is the first protein which was found to contain two RNA binding domains at the amino-terminus and a PPIase domain at the carboxyl-terminus. We isolated the hCyP33 gene from the human hematopoietic stem/progenitor cells and expressed it in Escherichia coli, and determined the crystal structure of the C domain of hCyP33 at 1.88 A resolution. The core structure is a beta-barrel covered by two alpha-helices. Superposition of the structure of the C domain of hCyP33 with the structure of CypA suggests that the C domain contains PPIase active site which binds to CsA. Furthermore, C domain seems to be able to bind with the Gag-encoded capsid (CA) of HIV-1 and may affect the viral replication of HIV-1. A key residue of the active site is changed from Ala-103-CypA to Ser-239-hCyP33, which may affect the PPIase domain/substrates interactions.     

Functional characterization of two novel parvulins in Trypanosoma brucei

Parvulins belong to a family of peptidyl-prolyl cis/trans isomerases (PPIases) that catalyze the cis/trans conformations of prolyl-peptidyl bonds. Herein, we characterized two novel parvulins, TbPIN1 and TbPAR42, in Trypanosoma brucei. TbPIN1, a 115 amino-acid protein, contains a single PPIase domain but lacks the N-terminal WW domain. Using NMR spectroscopy, TbPIN1 was found to exhibit PPIase activity toward a phosphorylated substrate. Overexpression of TbPIN1 can rescue the impaired temperature-sensitive phenotype in a mutant yeast strain. TbPAR42, containing 383 amino acids, comprises a novel FHA domain at its N terminus and a C-terminal PPIase domain but is a non-Pin1-type PPIase. Functionally, a knockdown of TbPAR42 in its procyclic form results in reduced proliferation rates suggesting an important role in cell growth.     

Chaperone-like activity of peptidyl-prolyl cis-trans isomerase during creatine kinase refolding

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 microM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.     

The peptidyl-prolyl isomerase and chaperone Par27 of Bordetella pertussis as the prototype for a new group of parvulins

Proteins that pass through the periplasm in an unfolded state are highly sensitive to proteolysis and aggregation and, therefore, often require protection by chaperone-like proteins. The periplasm of Gram-negative bacteria is well equipped with ATP-independent chaperones and folding catalysts, including peptidyl-prolyl isomerases (PPIases). The filamentous hemagglutinin of Bordetella pertussis, which is secreted by the two-partner secretion pathway, crosses the periplasm in an unfolded conformation. By affinity chromatography, we identified a new periplasmic PPIase of the parvulin family, Par27, which binds to an unfolded filamentous hemagglutinin fragment. Par27 differs from previously characterized bacterial and eukaryotic parvulins. Its central parvulin-like domain is flanked by atypical N- and C-terminal extensions that are found in a number of putative PPIases present mostly in β proteobacteria. Par27 displays both PPIase and chaperone activities in vitro. In vivo, Par27 might function as a general periplasmic chaperone in B. pertussis.     

Crystal Structure Determination and Functional Characterization of the Metallochaperone SlyD from Thermus thermophilus

SlyD (sensitive to lysis D; product of the slyD gene) is a prolyl isomerase [peptidyl-prolyl cis/trans isomerase (PPIase)] of the FK506 binding protein (FKBP) type with chaperone properties. X-ray structures derived from three different crystal forms reveal that SlyD from Thermus thermophilus consists of two domains representing two functional units. PPIase activity is located in a typical FKBP domain, whereas chaperone function is associated with the autonomously folded insert-in-flap (IF) domain. The two isolated domains are stable and functional in solution, but the presence of the IF domain increases the PPIase catalytic efficiency of the FKBP domain by 2 orders of magnitude, suggesting that the two domains act synergistically to assist the folding of polypeptide chains. The substrate binding surface of SlyD from T. thermophilus was mapped by NMR chemical shift perturbations to hydrophobic residues of the IF domain, which exhibits significantly reduced thermodynamic stability according to NMR hydrogen/deuterium exchange and fluorescence equilibrium transition experiments. Based on structural homologies, we hypothesize that this is due to the absence of a stabilizing β-strand, suggesting in turn a mechanism for chaperone activity by ‘donor-strand complementation.’ Furthermore, we identified a conserved metal (Ni²⁺) binding site at the C-terminal SlyD-specific helical appendix of the FKBP domain, which may play a role in metalloprotein assembly.     

Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli

The nature of molecular chaperones in the periplasm of Escherichia coli that assist newly translocated proteins to reach their native state has remained poorly defined. Here, we show that FkpA, a heat shock periplasmic peptidyl-prolyl cis/trans isomerase (PPIase), suppresses the formation of inclusion bodies from a defective-folding variant of the maltose-binding protein, MalE31. This chaperone-like activity of FkpA, which is independent of its PPIase activity, requires a full-length structure of the protein. In vitro, FkpA does not catalyse a slow rate-limiting step in the refolding of MalE31, but prevents its aggregation at stoichiometric amounts and promotes the reactivation of denaturated citrate synthase. We propose that FkpA functions as a chaperone for envelope proteins in the bacterial periplasm.     

Binding analysis of a psychrotrophic FKBP22 to a folding intermediate of protein using surface plasmon resonance

SIB1 FKBP22 is a homodimer, with each subunit consisting of the C-terminal catalytic domain and N-terminal dimerization domain. This protein exhibits peptidyl prolyl cis-trans isomerase activity for both peptide and protein substrates. However, truncation of the N-terminal domain greatly reduces the activity only for a protein substrate. Using surface plasmon resonance, we showed that SIB1 FKBP22 loses the binding ability to a folding intermediate of protein upon truncation of the N-terminal domain but does not lose it upon truncation of the C-terminal domain. We propose that the binding site of SIB1 FKBP22 to a protein substrate of PPIase is located at the N-terminal domain.     

NMR solution structure and dynamics of the peptidyl-prolyl cis-trans isomerase domain of the trigger factor from Mycoplasma genitalium compared to FK506-binding protein

We have solved the solution structure of the peptidyl-prolyl cis-trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy. Our results lead to a well-defined structure with a backbone rmsd of 0.23 A. As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold. Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar. Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the beta sheet. Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor. Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central beta sheet. Our data indicate, however, that this strand may be partially structured as "nascent" beta strand. This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases.     

PPIase domain of trigger factor acts as auxiliary chaperone site to assist the folding of protein substrates bound to the crevice of trigger factor

Trigger factor (TF) is the first chaperone encountered by nascent chains in bacteria, which consists of two modules: peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and a crevice built by both N- and C-terminal domains. While the crevice is suggested to provide a protective space over the peptide exit site of ribosome for nascent polypeptides to fold, it remains unclear whether PPIase domain is directly involved in assisting protein folding. Here, we introduced structural change into different regions of TF, and investigated their influence on the chaperone function of TF in assisting the folding of various substrate proteins, including oligomeric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monomeric carbonic anhydrase II (CA II) and lysozyme. Results showed that structural disturbances by site-specific mutations in the PPIase active site or by deletion of the PPIase domain from TF affected the chaperone activity of TF toward CA II and GAPDH but had no effect on TF-assisted lysozyme refolding, suggesting PPIase domain is involved in assisting the folding of substrates larger than lysozyme. Mutants with the structural disturbances in the crevice totally lost the chaperone activity toward all the substrates we used in this investigation. These results provide further evidence to confirm that the crevice is the major chaperone site of TF, and the hydrophobic pocket in PPIase domain acts as an auxiliary site to assist the folding of substrate proteins bound to the crevice in a substrate-dependent manner, which is beneficial for TF to provide appropriate assistance for protein folding by changing protective space and binding affinity.     

The Role of SurA PPIase Domains in Preventing Aggregation of the Outer-Membrane Proteins tOmpA and OmpT

SurA is a conserved ATP-independent periplasmic chaperone involved in the biogenesis of outer-membrane proteins (OMPs). Escherichia coli SurA has a core domain and two peptidylprolyl isomerase (PPIase) domains, the role(s) of which remain unresolved. Here we show that while SurA homologues in early proteobacteria typically contain one or no PPIase domains, the presence of two PPIase domains is common in SurA in later proteobacteria, implying an evolutionary advantage for this domain architecture. Bioinformatics analysis of > 350,000 OMP sequences showed that their length, hydrophobicity and aggregation propensity are similar across the proteobacterial classes, ruling out a simple correlation between SurA domain architecture and these properties of OMP sequences. To investigate the role of the PPIase domains in SurA activity, we deleted one or both PPIase domains from E. coli SurA and investigated the ability of the resulting proteins to bind and prevent the aggregation of tOmpA (19 kDa) and OmpT (33 kDa). The results show that wild-type SurA inhibits the aggregation of both OMPs, as do the cytoplasmic OMP chaperones trigger factor and SecB. However, while the ability of SurA to bind and prevent tOmpA aggregation does not depend on its PPIase domains, deletion of even a single PPIase domain ablates the ability of SurA to prevent OmpT aggregation. The results demonstrate that the core domain of SurA endows its generic chaperone ability, while the presence of PPIase domains enhances its chaperone activity for specific OMPs, suggesting one reason for the conservation of multiple PPIase domains in SurA in proteobacteria.     

On the benefit of bivalency in peptide ligand/pin1 interactions

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO₃H₂)/Thr(PO₃H₂)-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.     

Profound redox sensitivity of peptidyl-prolyl isomerase activity in Arabidopsis thylakoid lumen

Proteomic, enzymatic, and mutant analyses revealed that peptidyl-prolyl isomerase (PPIase) activity in the chloroplast thylakoid lumen of Arabidopsis is determined by two immunophilins: AtCYP20-2 and AtFKBP13. These two enzymes are responsible for PPIase activity in both soluble and membrane-associated fractions of thylakoid lumen suggesting that other lumenal immunophilins are not active towards the peptide substrates. In thiol-reducing conditions PPIase activity of the isolated AtFKBP13 and of the total thylakoid lumen is suppressed several fold. Profound redox-dependence of PPIase activity implies oxidative activation of protein folding catalysis under oxidative stress and photosynthetic oxygen production in the thylakoid lumen of plant chloroplasts.     

The SurA periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone activity

The Escherichia coli periplasmic peptidyl-prolyl isomerase (PPIase) SurA is involved in the maturation of outer membrane porins. SurA consists of a substantial N-terminal region, two iterative parvulin-like domains and a C-terminal tail. Here we show that a variant of SurA lacking both parvulin-like domains exhibits a PPIase-independent chaperone-like activity in vitro and almost completely complements the in vivo function of intact SurA. SurA interacts preferentially (>50-fold) with in vitro synthesized porins over other similarly sized proteins, leading us to suggest that the chaperone-like function of SurA preferentially facilitates maturation of outer membrane proteins.     

FKBP22 is part of chaperone/folding catalyst complexes in the endoplasmic reticulum of Neurospora crassa

FKBP22 is a dimeric protein in the lumen of the endoplasmic reticulum, which exhibits a chaperone as well as a PPIase activity. It binds via its FK506 binding protein (FKBP) domain directly to the Hsp70 chaperone BiP that stimulates the chaperone activity of FKBP22. Here we demonstrate additionally the association of FKBP22 with the molecular chaperones and folding catalysts Grp170, alpha-subunit of glucosidase II, PDI, ERp38, and CyP23. These proteins are associated with FKBP22 in at least two protein complexes. Furthermore, we report an essential role for FKBP22 in the development of microconidiophores in Neurospora crassa.     

Localization of the chaperone domain of FKBP52

FKBP52, a multidomain peptidyl prolyl cis/trans-isomerase (PPIase), is found in complex with the chaperone Hsp90 and the co-chaperone p23. It displays both PPIase and chaperone activity in vitro. To localize these two activities to specific regions of the protein, we created and analyzed a set of fragments of FKBP52. The PPIase activity toward both peptides and proteins is confined entirely to domain 1 (amino acids 1-148). The chaperone activity, however, resides in the C-terminal part of FKBP52, mainly in the region between amino acids 264 and 400 (domain 3). Interestingly, this domain also contains the tetratricopeptide repeats, which are responsible for the binding to C-terminal amino acids of Hsp90. Competition assays with a C-terminal Hsp90 peptide suggest that the non-native protein and Hsp90 are bound by different regions within this domain.     

FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli

The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.     

Prolyl isomerases in a minimal cell. Catalysis of protein folding by trigger factor from Mycoplasma genitalium.

Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the isomerization of prolyl peptide bonds. Distinct families of this class of enzymes are involved in protein folding in vitro, whereas their significance in free living organisms is not known. Previously, we inspected the smallest known genome of a self-replicating organism and found that Mycoplasma genitalium is devoid of all known PPIases except the trigger factor. Despite the extensive sequence information becoming available, most genes remain hypothetical and enzyme activities in many species have not been assigned to an open reading frame. Therefore, we studied the PPIase activity in crude extracts of M. genitalium. We showed that this is solely attributed to a single enzyme activity, the trigger factor. Characterization of this enzyme revealed that its PPIase activity resides in a central 12-kDa domain. Only the complete trigger factor is able to cis/trans isomerize extended peptide substrates, while the PPIase domain alone can not. The N- and the C-terminal domains of the trigger factor seem to function in binding of proteins as substrates, as demonstrated by protein refolding experiments, in which the complete trigger factor catalyzed protein refolding towards a model protein 500-fold more efficiently than the isolated central PPIase domain. Protein modeling studies suggest that the PPIase domain can fold in a similar way as the PPIase domain of FK506 binding proteins (FKBPs), one class of PPIases, despite only very limited sequence homology. Differences at the active site explain why this enzyme is not inhibited by FK506 in contrast with FKBPs. Trigger factor expressed in Escherichia coli confirms its additional chaperone functions, as shown by its association with chaperones GroEL and GroES after induction of misfolding. In contrast, the isolated PPIase-domain lacks any association with chaperones from E. coli. In summary, trigger factor of M. genitalium is the single folding isomerase of this organism, which harbors an enzymatically active PPIase domain with structural homology to FKBPs. Its additional domains confer its ability to be an efficient catalyst of protein folding. The protein folding machinery is conserved and shows a dual function as a chaperone and a prolyl isomerase.     

Comparative analysis of enzyme activities and mRNA levels of peptidyl prolyl cis/trans isomerases in various organs of wild type and Pin1-/- mice

We investigated the enzyme activity of peptidyl prolyl cis/trans isomerases (PPIases) in brain, testis, lung, liver, and mouse embryonic fibroblasts (MEF) of Pin1+/+ and Pin1-/- mice. The aim of this study is to determine if other PPIases can substitute for the loss of Pin1 activity in Pin1-/- mice and what influence Pin1 depletion has on the activities of other PPIases members. The results show that high PPIase activities of Pin1 are found in organs that have the tendency to develop Pin1 knockout phenotypes and, therefore, provide for the first time an enzymological basis for these observations. Furthermore we determined the specific activity (k(cat)/K(M)) of endogenous Pin1 and found that it is strongly reduced as compared with the recombinant protein in all investigated organs. These results suggest that posttranslational modifications may influence the PPIase activity in vivo. The activities originating from cyclophilin and FKBP are not influenced by the Pin1 knockout, but a basal enzymatic activity towards phosphorylated substrates could be found in Pin1-/- lysates. Real time PCR experiments of all PPIases in different mouse organs and MEF of Pin1+/+ and Pin1-/- mice support the finding and reveal the specific expression profiles of PPIases in mice.